Process for the preparation of a vaccine for the treatment of tuberculosis and other intracellular infections diseases and the vaccine produced by the process

ABSTRACT

The present invention relates to a process for the preparation of a vaccine against tuberculosis and other intracellular pathogens, this vaccine is targeted against intracellular pathogens, more particularly the pathogen  Mycobacterium tuberculosis  and Salmonella in this case.

FIELD OF INVENTION

The present invention relates to a process for the preparation of avaccine against tuberculosis and other intracellular pathogens. Thisvaccine is targeted against intracellular pathogens, more particularlythe pathogen Mycobacterium tuberculosis and Salmonella in this case.

The utility of the present invention is to develop a vaccine against theintracellular pathogens, which are causative agents of tuberculosis,brucellosis, leishmaniasis, listeriosis, leprosy, malaria, typhoid,trypanosomiasis and streptococcus and HIV-infection. The pathogenMycobacterium tuberculosis (M. tuberculosis) the subject matter of thisinvention is a causative agent of tuberculosis. In this invention M.tuberculosis was allowed to grow in the allogeneic and syngeneicmacrophages and macrophage cell lines. The macrophages—M. tuberculosiscomplex was then irradiated to kill the macrophages as well as themycobacterium.

BACKGROUND OF THE INVENTION

Tuberculosis is a chronic infectious disease that continues to kill some3 million people a year. About 8 million new cases arise every year andthe number continues to increase. About one-third of the worldpopulation is infected with M. tuberculosis. The emergence of AIDS hasreactivated tuberculosis in millions of dormant individuals, causing asharp rise in the number of cases and deaths. M. tuberculosis istherefore responsible for the highest morbidity rate among allinfectious agents. The only available vaccine BCG is both unpredictableand highly variable. Doubtful efficacy of BCG vaccination has put thescientific community to urgently develop effective means of vaccinationagainst the M. tuberculosis (Bloom, B. R. et. al., Annu. Rev. Immunol.10:1992:453).

During the past many decades BCG has been extensively used as a vaccineworld over. Several hundred million children and new born have been therecipient of BCG vaccine. However, in spite of wide usage of BCGvaccine, tuberculosis has still become the fastest spreading disease notonly in developing countries but also in the industrialized world.Further, the protective efficacy of the current BCG vaccine is bothunpredictable and highly variable and it remains the most controversialof all currently used vaccines. Its doubtful efficacy in controlledtrials have increased the concern about its use as a vaccine (Bloom andFine, Tuberculosis In B. Bloom (ed.), 1994:531, Bloom, B. R. et. al.,Annu. Rev. Immunol. 10:1992:453). Furthermore, the extensive clinicaltrials done in Madras showed similar extent of protection inBCG-vaccinated and unvaccinated individuals, indicating that BCG inducedzero protection (Ind. J. Med. Res. 1980:72(Suppl.):1-74). Thus it isobvious that BCG vaccination does not prevent transmission.

In past also, many questions always arose pertaining to the safe use ofBCG vaccine.

A major catastrophe that cast a cloud over the reputation of BCGvaccination occurred in 1929. In Lubeck, Germany, 251 children receiveda BCG vaccine prepared at a local institute, and 72 of these childrendied. Subsequent investigation revealed that the institute alsomaintained cultures of virulent tubercle bacilli and that the batch ofBCG vaccine given to the children had accidentally been contaminatedwith one of these strains of Mycobacterium tuberculosis (Lubeck. 1935.Die Sauglingstuberkulose in Lubeck. Springer, Berlin).

A new question has arisen regarding the safety of BCG in HIV-infectedindividuals. A small number of cases of disseminated BCG-osis have beenreported among children who received BCG vaccine and were subsequentlyfound to be HIV seropositive (Von Reyn, et. al. Lancet 1987: ii:669-672;Braun, et. al., Pedietr. Infect. Dis. J. 1992:11:220-227; Weltman, et.al., AIDS 7:1993:149). WHO currently recommends discontinuing the use ofBCG vaccine in children showing overt signs of immunodeficiency (WorldHealth Organization, 1992, Expanded Program for Immunization, ProgramReport,World Health Organization, Geneva; Weekly Epidemiol. Rec.62:1987:53).

Large volunteer studies by Dahlstrom and Difts (Scand J Respir DisSuppl. 65:1968:35) and a meta-analysis of BCG in the prevention oftuberculosis based on 13 prospective studies and 10 case control studieshas recently been completed (Colditz et. al., J. Amer. Med. Assoc.271:1994:698-702). While it concluded that on average BCG was about 50%protective in preventing tuberculosis, the biological and operationalsignificance of averaging, in essence, such widely divergent results areitself arguable.

Before the advent of AIDS, in most wealthy countries, the incidence oftuberculosis was declining for at least a century. This is illustratedin comparisons between The Netherlands (which never employed BCGvaccination) and the United Kingdom and Scandinavia (which institutednational BCG vaccination in the 1950). The declines in tuberculosiscases reported in these countries were similar (Styblo, K., SelectedPapers R. Netherland Tuberc. Assoc. 24:1991:136; Sutherland, Bull. Int.Union. 57:1981:17). Thus it is unreasonable to attribute that thedecline was due to BCG vaccination alone.

BCG's performance is based on a hypothesis that BCG is effective againstprimary infection in children and endogenous reactivation oflong-standing infections but not against exogenous infection (ten Dam,H. G. Adv. Tuberc. Res. 21:1984:79; ten Dam, H. G. and A. Pio. Tubercle63:1988:226). Epidemiological data suggest that BCG vaccination impartsgreater or more consistent protection against systemic disease, inparticular miliary tuberculosis and tuberculosis meningitis in children,than against pulmonary disease (Rodrigues, et.al., Int J epidemiol.22:1993:1154). Lurie's studies indicated that the number of CFU of M.tuberculosis isolated from lungs of BCG-immunized versus unimmunizedrabbits showed no difference in the number of organisms reaching andcapable of being cultured from lung and other tissues.

Another insight is provided by the intracellular location of themycobacterium. Electron microscopic findings indicate that BCG remainsessentially entirely within the phagolysosomes after in vitro infectionof macrophages, whereas virulent M. tuberculosis (strain H37Rv) canescape from the phagolysosome and enter the cytoplasm (McDonough,et.al., Infect. Immun. 61:1993:2763). This may be relevant insofar as itis the antigens in the endosomal compartment of antigen-presenting cellsthat are presented in conjunction with MHC class II determinants to CD4⁺T helper cells, whereas cytoplasmic antigens are presented inassociation with the Major Histocompatibility Complex (MHC) class Ideterminants to CD8⁺ Cytotoxic T cells (CTL). If these findings in vitroare general, they will explain why M. tuberculosis is more dependent forits elimination on MHC class I-restricted CTL than BCG and suggests thatBCG may not be very effective in eliciting MHC class I-restricted CTL(Stover, et.al., Nature 351:1991:456). In this context, Rich, 1951 (ThePathogenesis of Tuberculosis, 2^(nd), p. 1028; Charles C Thomas,Publisher, Springfield, Ill.), Canetti, 1955 (The Tubercule Bacilli inthe Pulmonary Lesion of Man, p. 226; Springer, N.Y.) and Lurie, 1964(Resistance to Tuberculosis. Experimental Studies in Native and AcquiredDefense, p. 391; Harvard University Press, Cambridge Press, Cambridge,Mass.), commented that recovery from infection with M. tuberculosisprovided stronger protection against future tuberculosis than could BCG.

The effective resistance to M. tuberculosis infection will requireparticipation both of specific CD8⁺ CTL to lyse macrophages orparenchymal cells unable to restrict their infection and of specificCD4⁺ T cells able to produce IL-2, IFN-γ, TNF-α, and other lymphokinesinvolved in macrophage activation.

Considering these drawbacks of the BCG-vaccine, the applicants havetaken advantage of the fact that the vaccine will be used as anirradiated preparation and has no fear of inoculating in AIDS patientsand immunocompromised children. BCG is given as an attenuatedpreparation and is not recommended in these subjects because it causesdisseminated BCG-osis, WHO currently recommends discontinuing the use ofBCG vaccine in children showing overt signs of immunodeficiency (WorldHealth Organization. 1992. Expanded Program for Immunization. ProgramReport. World Health Organization, Geneva. World Health Organization.Weekly Epidemiol. Rec. 1987:62:53-54).

Another insight is provided by the intracellular location of themycobacterium. BCG remains essentially entirely within the phagolysosomeof macrophages, whereas virulent M. tuberculosis can escape from thephagolysosome and enter the cytoplasm (McDonough, K. A., Y. Kress, andB. R. Bloom. 1993. Infect Immun. 61:2763-2773). The antigens in theendosomal compartment of antigen-presenting cells are presented inconjunction with MHC class II determinants to CD4⁺ T helper cells,whereas cytoplasmic antigens are presented in association with the MajorHistocompatibility Complex (MHC) class 1 determinants to CD8⁺ CytotoxicT cells. M. tuberculosis is more dependent for its elimination on MHCclass I-restricted CTL. BCG is not effective in eliciting MHC classI-restricted CTL (Stover, et.al., Nature 351:1991:456). The presentvaccine contains the irradiated preparation of M. tuberculosis grown inmacrophages. M. tuberculosis infected macrophages are reported toeffectively generate CTL (Stover, et.al., Nature 351:1991:456). Further,it has also been reported that irradiated cells undergo apoptosis andcan be phagocytosed by the dendritic cells (Albert, M. L., et.al.,Nature 392:1998:86) and it leads to the generation of antigen specificCD4⁺ and CD8⁺ T cell response. This apoptosis-dependent pathway may notonly have potential in vaccination studies but also for therapeuticallymanipulating immune system to induce T-helper and CTL response in vivoto a variety of antigens including tumor, and possibly to modulatefavourable immune response.

Rich (The pathogenesis of Tuberculosis. 2^(nd) ed, p. 1028, 1951.Charles C Thomas, Publisher, Springfield, Ill.), Canetti (The tubercleBacillus in the pulmonary Lesion of Man, p. 226, 1955. Springer, N.Y.),and Lurie (Resistance to Tuberculosis. Experimental Studies in Nativeand Acquired Defense, 391, 1964. Harvard University Press, Cambridge,Mass.) have commented that recovery from infection with M. tuberculosisprovided stronger protection against future tuberculosis than could BCG.In context with the above statements, the candidate vaccine hasadvantage over existing BCG vaccine because it contains the M.tuberculosis grown in the natural environment of the macrophages thatsecrete the unique antigens responsible for the induction of protectiveimmune response and can generate CD4⁺ T-helper cells and CD8⁺ CTL. Theeffective resistance to M. tuberculosis infection will requireparticipation of both specific CD8⁺ CTL to lyse macrophages orparenchymal cells unable to restrict their infection and of specificCD4⁺ T cells able to produce IL-2, IFN-γ, TNF-α, and other lymphokinesinvolved in macrophage activation.

The main rationale behind this process was to develop a vaccine againsttuberculosis and other intracellular diseases, MHC-matched (syngeneic)and mismatched (allogeneic) macrophages harboring M. tuberculosis onirradiation undergo apoptosis; dendritic cells engulf these macrophagesand present the antigen (Mycobacterium-proteins and allo-macrophagepeptides) on their surface and induce naïve T-cells to differentiateinto effecter CD4* Th1 cells. These dendritic cells also activate CD8* Tcells for cell-mediated immunity. Allo-macrophages in the systemgenerate an allo-reaction and as a result a large amount of cytokineslike IL-2, IL-12, IFN-γ, etc., are produced which promote the Th1response and cell mediated immune response. It is known that Th1-type ofresponse provides protection against tuberculosis. Hence the mainutility of the process was to produce a potent and specific vaccineagainst M. tuberculosis.

OBJECTS OF THE INVENTION

The main object of the present invention thus is to develop a vaccineagainst tuberculosis and other intracellular diseases like leprosy,leishmaniasis, typhoid, trypanosomiasis malaria, brucellosis,listeriosis, AIDS, streptococcal infection and cancer.

Another object of the present invention is to culture the pathogeninside the syngeneic and allogeneic macrophages and allow them tosecrete antigens within the cells.

Yet another object is to develop a method whereby the pathogen arekilled by the already known drugs and further will be gamma irradiatedbefore use; the gamma irradiated cells are known to undergo apoptosisand are engulfed by the dendritic cells. Dendritic cells are potentactivator of Th1 cells and CD8+ cytotoxic cells.

Another object is to develop a vaccine that acts against both syngeneicmacrophages entrapped pathogens (viz. M. tuberculosis, M. leprae,leishmania, salmonella, trypanosoma, malaria, brucella, listeria, HIV,streptococcus) (e.g. SMTV, S=syngeneic, M=macrophage, T=tuberculosis,V=vaccine) and allogeneic-macrophages entrapped pathogen vaccine (e.g.,AMTV, A=allo, M=macrophage, T=tuberculosis, V=vaccine), to generateprotective immune response.

Still another objective of the present invention is to develop a vaccinebased on entrapment of pathogen in the allogeneic cells that wouldelicit immune response irrespective of the genetic background i.e. itwill work as a promiscuous vaccine, and hence it will work irrespectiveof the genetic diversity in the human subjects.

SUMMARY OF THE INVENTION

The present invention relates to a process for the preparation of avaccine against tuberculosis and other intracellular pathogens. Thisvaccine is targeted against intracellular pathogens, more particularlythe pathogen Mycobacterium tuberculosis and Salmonella in this case.

DESCRIPTION OF THE ACCOMPANYING DRAWINGS

FIG. 1 represents schematically the process of how Allo-MacrophageTuberculosis (AMTV) works.

DETAILED DESCRIPTION OF THE INVENTION

The novelty in the present invention is that the protective antigenssecreted by the mycobacterium inside the macrophages can be used as avaccine without isolating them from the macrophages.

The vaccine was used after irradiation and the irradiated cells areknown to undergo apoptosis. The cells undergoing apoptosis were engulfedby the dendritic cells. Dendritic cells activated naïve T cells todifferentiate into Th1 cells and cytotoxic cells. These cells are knownto be cardinal in imparting protective immunity against intracellularinfections and cancer.

Allo-macrophages in the system generated allo-reaction as a result largeamount of cytokines like IL-2, IL-12, IFN-γ, etc., are produced whichpromote the Th1 response and cell mediated immune response. Theallogeneic cells used in the construction of vaccine would elicit immuneresponse irrespective of the genetic background i.e. it will work as apromiscuous vaccine. Hence it can used in human subjects irrespective ofthe genetic diversity.

The aim of the present invention is to develop a vaccine againsttuberculosis, salmonella and other intracellular infections. M.tuberculosis and Salmonella typhimurium was cultivated in allogeneic(AMTV) and syngeneic (SMTV) macrophages and was killed by γ-irradiationand was used as a vaccine. The AMTV in vivo will preferably be engulfedby dendritic cells (γ-irradiation causes cells to undergo apoptosis anddendritic cells engulf apoptotic cells) and will then activate themycobacterium reactive naïve T cells. Allogeneic macrophages being usedfor immunization worked as an adjuvant and elicited allogeneic reactiveT cells that produced huge amount of IL-2, IFN-γ, IL-12. These cytokinesare vital for the growth and differentiation of naïve T cells to CD4⁺and CD8⁺ effector T cells. Dendritic cells are the preferred AntigenPresenting Cells (APC) for Th1 and cytotoxic T cells (CTL). Th1 and CD8⁺CTL are principal cells in generating effective and protective immunityagainst M. tuberculosis. The tuberculosis resistant and susceptiblestrains of mice were inoculated with the vaccine.

FIG. 1 represents schematically the process of how Allo-MacrophageTuberculosis (AMTV) works.

The rationale behind the process of how Allo-Macrophage Tuberculosis(AMTV) works has been schematically shown in FIG. 1. M. tuberculosis wascultivated in MHC-mismatched (allogeneic) and syngeneic macrophages.This preparation was γ-irradiated and used as vaccine. The AMTV in vivowill preferably be engulfed by dendritic cells (as it is known thatγ-irradiation causes cells to undergo apoptosis and dendritic cellsengulf apoptotic cells) and will then activate the mycobacteriumreactive naïve T cells. However, macrophages loaded with mycobacteriumcannot activate naïve T cells directly. Allogeneic macrophages beingused for immunization would elicit allogeneic reactive T cells thatproduce huge amount of IL-2, IFN-γ, IL-12. These cytokines are vital forthe growth and differentiation of naïve T cells to CD4⁺ and CD8⁺effector T cells. Dendritic cells are the preferred Antigen PresentingCells (APC) for Th1 and cytotoxic T cells (CTL). They cause stimulationof naïve T cells to differentiate into antigen reactive Th1 cells andcytotoxic T lymphocytes. Moreover, dendritic cells trap foreign antigen(in this case mycobacterium antigen) and act as a reservoir, slowlyreleasing the antigen in the system for the activation of T cells andfor the maintenance of memory cells. IL-2, IFN-γ and IL-12 secreted byalloreactive T cells will engineer the clonal expansion of mycobacteriumreactive Th1 and cytotoxic T cells. Th1 and CD8⁺ CTL are cardinal ingenerating effective and protective immunity against M. tuberculosis.The tuberculosis resistant and susceptible strains of mice wereinoculated with the vaccine.

The rationale behind the process of how Allo-Macrophage Tuberculosis(AMTV) works has been demonstrated by cultivating M. tuberculosiscultivating in MHC-mismatched (allogeneic) and syngeneic macrophages.This preparation was γ-irradiated and used as vaccine. The AMTV in vivowill preferably be engulfed by dendritic cells (as it is known thatγ-irradiation causes cells to undergo apoptosis and dendritic cellsengulf apoptotic cells) and will then activate the mycobacteriumreactive naïve T cells. However, macrophages loaded with mycobacteriumcannot activate naïve T cells directly. Allo-macrophages being used forimmunization will elicit allo-reactive T cells that produce huge amountof IL-2, IFN-γ, IL-12. These cytokines are vital for the growth anddifferentiation of naïve T cells to CD4⁺ and CD8⁺ effector T cells.Dendritic cells are the preferred Antigen Presenting Cells (APC) for Th1and cytotoxic T cells (CTL). They cause stimulation of naïve T cells todifferentiate into antigen reactive Th1 cells and cytotoxic Tlymphocytes. Moreover, dendritic cells trap foreign antigen (in thiscase mycobacterium antigen) and act as a reservoir, slowly releasing theantigen in the system for the activation of T cells and for themaintenance of memory cells. IL-2, IFN-γ and IL-12 secreted byalloreactive T cells will engineer the clonal expansion of mycobacteriumreactive Th1 and cytotoxic T cells. Th1 and CTL are cardinal ingenerating effective and protective immunity against M. tuberculosis(Albert, M. L., et. al., Nature 392:1998:86; Wang, B. et. al., Proc.Natl. Acad. Sci. USA 90:1993:4156) The tuberculosis resistant andsusceptible strains of mice were vaccinated with AMTV and SMTV. Theefficacy of the vaccine was monitored by infecting the mice with live M.tuberculosis and monitoring their mortality and viable counts of thebacteria in the lungs, spleen and liver The vaccinated (4-12 weeks) micewere challenged with 10⁵-10⁶ viable M. tuberculosis H37Rv. The lungs,spleens and livers of the infected mice were removed after an additionalperiod of 3-4 weeks and serial dilutions of organ homogenate was platedon agar plates to establish the number of viable tubercle bacilliresiding in these organs. The vaccinated animals were also monitored forthe generation of Th1 and Th2 cells by measuring IFN-γ and IL-4. Thevaccine was inoculated in the mouse footpad and the induction of delayedtype hypersensitivity reaction was monitored by measuring the thicknessof the footpad.

According to the present invention there is provided a novel vaccineagainst tuberculosis and other intracellular pathogens and a process forthe development thereof. The tuberculosis vaccine (SMTV and AMTV),comprise M. tuberculosis cultivated in MHC-matched andmismatched-macrophages. The preparations are irradiated and used asdistinct vaccines.

Since the vaccine fulfill all the requirements necessary for generatingfavourable immune response against M. tuberculosis, it has beenanticipated that such preparations should work effectively againsttuberculosis.

The vaccine AMTV works in a promiscuous manner, since it does not followthe rules of MHC-restriction and is based on allo-stimulation andengulfment of foreign-apoptotic cells by dendritic cells. Whereas thevaccine SMTV works in MHC-restriction fashion.

The infected cells were grown in sufficient quantity and stored afterisoniazid treatment and γ-irradiation. The preparation was thoroughlychecked for viable mycobacterium by viability counting. None of thebacteria were viable in the vaccine. The mice were vaccinatedintraperitoneally or subcutaneously with vaccine and were challengedwith viable M. tuberculosis H37Rv. The viability of the tubercle bacilliresiding in lungs, spleens and livers was monitored. The animals wereimmunized with the vaccine and the uptake of the apoptotic cells bydendritic cells was documented by immunofluorescence. The animals werevaccinated with SMTV and AMTV and the proliferation and differentiationof naïve CD4⁺ Th cells into effector Th1 and Th2 subtype was studied. Asa control, M. tuberculosis entrapped in syngeneic macrophages was alsoused. The ability of SMTV and AMTV to generate CD8⁺ cytotoxic T cellswas monitored by the standard Cr⁵¹-release assay.

To test the hypothesis of allo-stimulation, Balb/c (IA^(d)) and C57BL/6(IA^(b)) strains of mice were immunized with ovalbumin entrapped inmitomycin C treated allogeneic and syngeneic APC. To eliminate thepossibility of preferably generating allo-response in secondaryresponse, the haplotype of the allo-APC was changed. The animals weregiven secondary booster with ovalbumin entrapped in the APC of CBA(IA^(k)) mice. Profound activation of CD4⁺ and CD8⁺ T cells wasobserved. Antigen-specific-T cell proliferation and predominant Th1response were noticed, as evidenced by mainly the production of IL-2 andIFN-γ and IgG2a-isotype. High production of IL-2 in allo-response wasnoticed which indicates that the immunization with the antigen entrappedin allo-APC treated with mitomycin C undergoes apoptosis. The apoptoticcells are engulfed by dendritic cells that then evokes mycobacteriumspecific and the allo-reactive T cells response. The allo-T cellsare >10% of the total T cell population and are known to induce highsecretion of IL-2. IL-2 produced by allo-T cells then engineers theproliferation of antigen specific T cell.

Therefore, in the present invention the development of effectivetuberculosis vaccine; based on a novel delivery system targeted todendritic cells, M. tuberculosis was cultivated in the macrophage cellline viz. J77.4 or allogeneic and syngeneic macrophages. The infectedmacrophages were isoniazid treated and irradiated and then used forvaccination studies in protection against M. tuberculosis.

The rationale behind the process of how Allo-Macrophage Tuberculosis(AMTV) works has been demonstrated by cultivating M. tuberculosiscultivating in MHC-mismatched (allogeneic) and syngeneic macrophages.This preparation was γ-irradiated and used as vaccine. The AMTV in vivowill preferably be engulfed by dendritic cells (as it is known thatγ-irradiation causes cells to undergo apoptosis and dendritic cellsengulf apoptotic cells) and will then activate the mycobacteriumreactive naïve T cells. However, macrophages loaded with mycobacteriumcannot activate naïve T cells directly. Allo-macrophages being used forimmunization will elicit allo-reactive T cells that produce huge amountof IL-2, IFN-γ, IL-12. These cytokines are vital for the growth anddifferentiation of naïve T cells to CD4⁺ and CD8⁺ effector T cells.Dendritic cells are the preferred Antigen Presenting Cells (APC) for Th1and cytotoxic T cells (CTL). They cause stimulation of naïve T cells todifferentiate into antigen reactive Th1 cells and cytotoxic Tlymphocytes. Moreover, dendritic cells trap foreign antigen (in thiscase mycobacterium antigen) and act as a reservoir, slowly releasing theantigen in the system for the activation of T cells and for themaintenance of memory cells. IL-2, IFN-γ and IL-12 secreted byalloreactive T cells will engineer the clonal expansion of mycobacteriumreactive Th1 and cytotoxic T cells. Th1 and CTL are cardinal ingenerating effective and protective immunity against M. tuberculosis(Albert, M. L., et. al., Nature 392:1998:86; Wang, B. et. al., Proc.Natl. Acad. Sci. USA 90:1993:4156). The tuberculosis resistant andsusceptible strains of mice were vaccinated with AMTV and SMTV. Theefficacy of the vaccine was monitored by infecting the mice with live M.tuberculosis and monitoring their mortality and viable counts of thebacteria in the lungs, spleen and liver. The vaccinated (4-12 weeks)mice were challenged with 10⁵-10⁶ viable M. tuberculosis H37Rv. Thelungs, spleens and livers of the infected mice were removed after anadditional period of 3-4 weeks and serial dilutions of organ homogenatewas plated on agar plates to establish the number of viable tuberclebacilli residing in these organs. The vaccinated animals were alsomonitored for the generation of Th1 and Th2 cells by measuring IFN-γ andIL-4. The vaccine was inoculated in the mouse footpad and the inductionof delayed type hypersensitivity reaction was monitored by measuring thethickness of the footpad.

Accordingly, the present invention provides a vaccine againsttuberculosis and other intracellular pathogens selected from the groupconsisting of Mycobacterium leprae, leishmania, salmonella, typanosoma,plesmodium, brucella, listeria, HIV, streptococcus and cancer. Theinvention also provides a method for the development of the saidvaccine, comprising the steps of:

(i) culturing pathogens selected from the group comprising Mycobactenumtuberculosis, Mycobactenum leprae, leishmania, salmonella, typanosoma,plasmodium, brucella, listeria, HIV, and streptococcus;

(ii) culturing syngeneic (same strain), allogeneic (different strain)and xenogeneic (different species like sheep and goat) macrophages andmacrophage cell lines selected from the group consisting of J774,P388D1, RAW, BMC-2, THP-1, etc.;

(iii) infecting macrophages and cell lines with a pathogen;

(iv) treating the infected cells with known drugs followed by gammairradiation to obtain the vaccine;

(v) immunizing disease resistant and susceptible strains of animals withthe vaccine obtained above;

(vi) infecting the animals with live pathogen and monitoring theirmortality and viable counts of infectious agent in lungs, spleen andliver; and

(vii) monitoring the vaccinated animals for proliferation and generationof CD4* Th1 and Th2 cells and CD8* cytotoxic T cells indicating thegeneration of cell mediated immunity.

The invention further provides a process for the preparation of avaccine against tuberculosis, wherein the said process comprising thesteps of:

(i) culturing of Mycobacterium tuberculosis H37Rv;

(ii) culturing of syngeneic and allogeneic macrophages and macrophagecell lines selected from the group consisting of J774, P388D1, RAW,BMC-2, THP-1, etc.;

(iii) infecting macrophages and cell lines (J774, P388D1, RAW, BMC-2,THP-1) with M. tuberculosis;

(iv) treating the infected cells with isoniazid and gamma irradiation toobtain the vaccine;

(v) immunizing tuberculosis resistant and susceptible strains of micewith allogeneic macrophage tuberculosis vaccine (AMTV) and syngeneicmacrophage tuberculosis vaccine (SMTV) obtained above;

(vi) infecting the mice with live M. tuberculosis and monitoring theirmortality and viable counts of bacteria in lungs, spleen and liver;

(vii) monitoring the vaccinated animals for proliferation and generationof CD4* Th1 and Th2 cells and CD8* cytotoxic T cells indicating thegeneration of cell mediated immunity; and

(viii) inoculating the vaccine in the mouse footpad and examining thedelayed type hypersensitivity reaction by measuring the swelling in thefootpad for protective immunity.

The invention also provides a process for the preparation of a vaccineagainst salmonella, wherein the said process comprising the steps of:

(i) culturing of Salmonella typhimurium;

(ii) culturing of syngeneic and allogeneic macrophages and macrophagecell lines selected from the group consisting of J774, P388D1, RAW,BMC-2, THP-1, etc.;

(iii) infecting macrophages and cell lines (J774, P388D1, RAW, BMC-2,THP-1) with S. typhimurium;

(iv) treating the infected cells with mitomycin C and gamma irradiationto obtain the vaccine;

(v) immunizing tuberculosis resistant and susceptible strains of micewith the vaccine obtained above;

(vi) infecting the mice with live S. typhimurium and monitoring theirmortality and viable counts of bacteria in lungs, spleen and liver;

(vii) monitoring the vaccinated animals for proliferation and generationof CD4* Th1 and Th2 cells and CD8* cytotoxic T cells indicating thegeneration of cell mediated immunity; and

(viii) inoculating the vaccine in the mouse footpad and examining thedelayed type hypersensitivity reaction by measuring the swelling in thefootpad for protective immunity.

The invention provides a vaccine by entrapment of M. tuberculosis,Salmonella and other intracellular pathogens in the allogeneic andsyngeneic macrophages and using it for the protection against theinfectious agent.

The process of the present invention is illustrated in the examplesgiven below which should not, however, be constructed to limit the scopeof the present invention.

EXAMPLE 1 A Process for the Preparation of a Vaccine AgainstTuberculosis and Other Intracellular Pathogens

The intracellular pathogens viz. Mycobacterium tuberculosis,Mycobacterium leprae, leishmania, salmonella, trypanosoma, plasmodium,brucella, listeria, HIV, streptococcus were cultured in the macrophagesof syngeneic and allogeneic mice, macrophages cell lines J774, P338D1,RAW, BMC-2, THP-1 (ATCC, Rockville). The infected cells were treatedwith isoniazid (20 μg/ml) for 48h at 37° C./5% CO₂ and irradiated at0.05 kGy.

(i).

(a) The resultant infected cells were treated with the pathogen specificdrug and further irradiated and was used as a vaccine and their efficacywas monitored by challenging the vaccinated mice with the viablebacteria. The efficacy of the vaccine was monitored by counting theviability of the infectious organism in the lungs, spleens and livers ofthe infected mice by serial dilutions of organ homogenate plated on theagar plates after definite interval of time. Similarly, the unvaccinatedanimals were challenged with live bacteria and were monitored for theirmortality and viable counts in lungs, spleens and livers.

(b) The vaccinated animals were monitored for proliferation anddifferentiation of CD4⁺ Th cell into bacteria reactive effectorcytotoxic T cells, Th1 and Th2 cells by measuring IFN-γ and IL-4 byELISA.

(c) CD8⁺ cytotoxic T cells were monitored by ⁵¹Cr-release assay.

EXAMPLE 2 A Process for the Preparation of a Vaccine AgainstTuberculosis

In another example, Mycobacterium tuberculosis H37Rv obtained fromCentral JALMA Institute for Leprosy, Agra, was cultured in themacrophages of syngeneic and allogeneic mice, macrophages cell linesJ774, P338D1, RAW, BMC-2, THP-1 (ATCC, Rockville). The infected cellswere treated with isoniazid (20 μg/ml) for 48 h at 37° C./5% CO₂ andirradiated at 0.05 kGy.

(i).

(a) The resultant infected cells were treated with isoniazid and thengamma irradiated to use as a vaccine and their efficacy was monitored bychallenging the vaccinated mice with 10⁵-10⁶ viable bacteria. In thiscase the lungs, spleens and livers of the infected mice were removedafter an additional period of 3-4 weeks and serial dilutions of organhomogenate was plated on agar plates to establish the number of viableM. tuberculosis residing in the these organs. Similarly, theunvaccinated animals were challenged with live bacteria and weremonitored for their mortality and viable counts in lungs, spleens andlivers.

(b) The vaccinated animals were monitored for proliferation anddifferentiation of T cells into bacteria reactive effector CD8⁺cytotoxic T cells and CD4⁺ Th1 and Th2 cells by measuring IFN-γ and IL-4by ELISA.

(c) CD8⁺ cytotoxic T cells were monitored by ⁵¹Cr-release assay.

(d) The vaccine was inoculated in the mouse footpad and the delayed typehypersensitivity reaction was monitored by measuring the thickness ofthe footpad.

EXAMPLE 3 A Process for the Preparation of a Vaccine Against Salmonella

In another example, Salmonella typhimurium (MTCC98) was cultured in themacrophages obtained from syngeneic and allogeneic mice and macrophagescell lines J774, BMC-2 and RAW. The infected cells were treated withmitomycin C (50 μg/ml) and gamma irradiated (0.05 kGy).

(i).

(a) The resultant infected cells were treated with the drug andirradiated and were used as a vaccine and their efficacy was monitoredby challenging the vaccinated mice with 10⁵-10⁶ viable bacteria. Theanimals were observed for mortality for 21 days. The lungs, spleens andlivers of the infected mice were removed and serial dilutions of organhomogenates was plated on agar plates to establish the number of viablesalmonella bacilli residing in these organs. Similarly, the unvaccinatedanimals were challenged with live bacteria and were monitored for theirmortality and viable counts in lungs, spleens and livers.

(b) The vaccinated animals were monitored for proliferation anddifferentiation of CD4⁺ Th cell into bacteria reactive effector Th1 andTh2 cells by measuring IFN-γ and IL-4 by ELISA.

(c) CD8⁺ cytotoxic T cells were monitored by ⁵¹Cr-release assay.

(d) The vaccine was inoculated in the mouse footpad and the delayed typehypersensitivity reaction was monitored by measuring the thickness ofthe footpad.

ADVANTAGES

The main advantages of the present invention are:

(i) About one-third of the world population is infected with M.tuberculosis. About 5-10% only develop active tuberculosis and the 90%of the individual develop effective immunity against the M.tuberculosis. M. tuberculosis present in the host macrophages secretesunique antigens, which are the effective inducers of long lastingprotective immunity. In contrast, M. tuberculosis when cultured in vitroin artificial medium, secrete antigens that do not induce optimum levelof protection and the immunity generated is short lived. The outstandingfeature in the process is that the protective antigens of mycobacteriumsecreted inside the macrophages were utilized without isolating themfrom the macrophages.

(ii) These allo-macrophages used in the system functions as a uniquesystem for delivering antigens secreted by live mycobacterium todendritic cells and as an adjuvant for eliciting the secretion ofcytokines viz. IL-2, IL-12, IFN-γ, etc., from allo-reactive T cells. Theexcess of IL-2 secreted was utilized by the mycobacterium reactiveprotective T cells. Therefore there was no need for any adjuvant to beused. Alloreactive T cells produce chiefly IL-2, IFN-γ and IL-12, thecytokines responsible for the generation of Th1-like immune response.Th1 are crucial for inducing protective immunity against M.tuberculosis.

(iii) The advantage of the invention is that the γ-irradiatedmycobacterium infected macrophages were engulfed by dendritic cells. Theγ-irradiated cells are known to undergo apoptosis. Apoptotic cells takenup by the dendritic cells induce the activation of CD4⁺ Th1 and CD8⁺cytotoxic T cells. Cytotoxic T cells are responsible for the killing ofmacrophages infected with mycobacterium. Lysis of target is essential inthe case of diseases like tuberculosis, typhoid, leprosy, leishmaniasis,AIDS, etc., where the pathogen reside and multiplies within themacrophages. Lysis of these cells liberates the pathogen and gives anopportunity to activated macrophages to engulf the bacteria andeliminate it. Dendritic cells express high level of B7-1 and secreteIL-12 and are only potent APC that can activate naïve T cells. Moreover,dendritic cells can differentiate naïve T cells to Th1 and CD8⁺cytotoxic T cells. Th1 and cytotoxic T cells are vital for induction ofprotective immunity against M. tuberculosis (Wakeham, et. al., J.Immunol., 160:1998:6101).

(iv) Another advantage and uniqueness in the invention is that thedendrites on dendritic cells trap the foreign antigens and work as areservoir. This antigen is slowly released from the dendrites, and isresponsible for the maintenance of memory cells.

(v) The AMTV vaccine works in MHC-unrestricted manner, because it isbased on allo-stimulation and engulfment of apoptotic cells by dendriticcells. It will work for all human, irrespective of the geneticdiversity.

What is claimed is:
 1. A process for the preparation of a vaccineagainst an intracellular pathogen selected from the group consisting ofM. tuberculosis, M. leprae, Leishmania, Salmonella, Trypanosoma,Plasmodium, Brucella, Listeria, and Streptococcus, wherein the processcomprises the steps of: (i) culturing the intracellular pathogen; (ii)culturing syngeneic (same strain), allogeneic (different strain) andxenogeneic (different species like sheep and goat) macrophages andmacrophage cell lines selected from the group consisting of J774,P388D1, RAW, BMC-2 and THP-1; (iii) infecting the macrophages andmacrophage cell lines with the selected pathogen of step (i); (iv)treating the infected macrophages and macrophage cell lines withpathogen specific drugs to kill the pathogen, followed by gammairradiation to kill the macrophage or macrophage cell line and remainingpathogens to obtain a composition; (v) immunizing disease resistant andsusceptible strains of animals with the composition; (vi) infecting thevaccinated animals with live selected pathogen and monitoring animalmortality, and viable counts of the pathogen in lungs, spleen and liver;(vii) monitoring the vaccinated animals for proliferation and generationof CD4* Th1 and Th2 cells and CD8* cytotoxic T cells indicating thegeneration of call mediated immunity against the pathogen; and (viii)wherein the composition is a vaccine if administration of thecomposition results in decreased mortality of vaccinated animals and/ordecreased viable counts of the pathogen in lungs, spleen, and liver ofthe infected animals when compared to non-immunized animals.
 2. Aprocess for the preparation of a vaccine against tuberculosis, theprocess comprising the steps of: (i) culturing M. tuberculosis H37Rv;(ii) culturing syngeneic, allogeneic and xenogenic macrophages andmacrophage cell lines selected from the group consisting of J774,P388D1, RAW, BMC-2 and THP-1; (iii) infecting the macrophages andmacrophage cell lines (J774, P388D1, RAW, BMC-2, THP-1) with M.tuberculosis; (iv) treating the infected macrophage and macrophage celllines with amikacin, isoniazid and gamma irradiation to kill themacrophage or macrophage cell line and remaining M. tuberculosis toobtain a composition; (v) immunizing tuberculosis resistant andsusceptible strains of mice with an allogeneic macrophage tuberculosiscomposition or syngeneic macrophage tuberculosis composition orxenogenic macrophages tuberculosis; (vi) infecting the vaccinated groupof mice with live M. tuberculosis and monitoring animal mortality, andviable counts of M. tuberculosis in lungs, spleen and liver; (vii)monitoring the vaccinated animals for proliferation and generation ofCD4* Th1 and Th2 cells, and CD8* cytotoxic T cells indicating thegeneration of cell mediated immunity against M. tuberculosis; and (viii)wherein the composition is a vaccine if administration of thecomposition results in decreased mortality of vaccinated animals and/ordecreased viable counts of M. tuberculosis in lungs, spleen, and liverof the infected animals when compared to non-immunized animals.
 3. Aprocess for the preparation of a vaccine against salmonella, the processcomprising the steps of: (i) culturing Salmonella typhimurium; (ii)culturing syngeneic, allogeneic macrophages and xenogenlc macrophagesand macrophage cell lines selected from the group consisting of J774,P388D1, RAW, BMC-2 and THP-1; (iii) infecting the macrophages andmacrophage cell lines (J774, P388D1, RAW, RMC-2, THP-1) with S.typhimurium; (iv) treating the macrophage and macrophage cell lines withmitomycin C and gamma irradiation to kill the macrophage or macrophagecell line and remaining S. typhimurium to obtain a composition; (v)immunizing salmonella resistant and susceptible strains of mice with thecomposition; (vi) infecting the vaccinated group of mice with live S.typhimurium and monitoring animal mortality, and viable counts of S.typhimurium in lungs, spleen and liver, (vii) monitoring the vaccinatedanimals for proliferation and generation of CD4* Th1 and Th2 cells, andCD8* cytotoxic T cells indicating the generation of cell mediatedimmunity against S. typhimurium; and (viii) wherein the composition is avaccine if administration of the composition results in decreasedmortality of vaccinated animals and/or decreased viable counts of S.typhimurium in lungs, spleen, and liver of the infected animals whencompared to non-immunized animals.
 4. A vaccine as prepared by theprocess as claimed in claim 1, wherein by entrapment of M. tuberculosis,Salmonella typhimurium and other intracellular pathogens in theallogeneic, syngeneic and xenogenic macrophages, the preparations beingtreated by the available drugs selected from amikacin, isoniazid andgamma radiation for tuberculosis and mitomycin C and gamma radiation forS. typhimurium to kill the pathogens and the vaccine is further gammairradiated before being used for the protection against the infectiousdiseases.